Before staining, warm slides at room temperature for 30
minutes and fix in ice cold acetone for 5 minutes. Air
dry for 30 minutes.

Wash in PBS

C.
Paraffin Sections

Deparaffinize sections in xylene, 2x5min.

Hydrate with 100% ethanol, 2x3min.

Hydrate with 95% ethanol, 1min.

Rinse in distilled water.

Follow procedure for pretreatment as required.

2. Pretreatments of Tissue Sections

Antigenic
determinants masked by formalin-fixation and paraffin-embedding
often may be exposed by epitope umasking, enzymatic digestion or
saponin, etc. Do not use this pretreatment
with frozen sections or cultured cells that are not
paraffin-embedded.

3. Procedure

Note:
prior to perform double labeling, it is important to test each
primary antibody individually and select the best pretreatment(s)
for each antibody. It will be ideal if the two primary antibodies
require same pretreatment. Otherwise, one should do a further test
by treating sections with both pretreatments and then immunostain
for each antibody individually. If both antibodies survive the
“double pretreatments”, you are ready for immunohistochemistry
double staining. Another alternative is to do pretreatments
separately for each antibody staining.

Rinse Sections in PBS-Tween 20 for 2x2
min.

Serum Blocking: incubate sections in normal serum
blocking solution – species same as secondary antibody (for
example: primary antibodies are mouse and rabbit, and secondary
antibodies are horse anti-mouse, and goat anti-rabbit, so horse
and goat serum block should be used).

Primary Antibodies: incubate
sections in the mixture of two primary antibodies (mouse
and rabbit) at appropriate dilution in
antibody dilution buffer
for 1 hour at room temperature.